Inotropic antibodies and therapeutic uses thereof

ABSTRACT

Antibodies binding to sites on the α-subunit (Na + +K + )-ATPase increase cardiac contraction of both ventricular myocytes and mouse heart. In particular, antibodies binding to the RSATEEEPPNDD or DVEDSYGQQWTYEQR peptides (or isoforms/derivatives thereof) of the α-subunit of the (Na + +K + )-ATPase, have been found to be highly inotropic. Both the antibodies and the peptides are important for the treatment of human heart failure and other contractile disorders.

[0001] This application claims the benefit of U.S. provisionalapplication No. 60/391,514 filed Jun. 25, 2002 and U.S. provisionalapplication No. 60/456,879 filed Mar. 21, 2003, both of which are herebyincorporated by reference herein in their entirety.

FIELD OF THE INVENTION

[0002] Structural regions of the (Na⁺+K⁺)-ATPase that are directlyinvolved in the regulation of cardiac contractility are provided.Site-specific binding of these antibodies against the known sequences ofthe (Na⁺+K⁺)-ATPase significantly increased cardiac contraction of bothisolated rat cardiac myocytes and mouse hearts. These important findingsprovide functional connection between the structure of the(Na⁺+K⁺)-ATPase and cardiac positive inotropy, wherein a novel mode for(Na⁺+K⁺)-ATPase to regulate cardiac function is shown.

BACKGROUND

[0003] (Na⁺+K⁺)-ATPase is a target receptor for digitalis and relateddrugs. Digitalis, digoxin, ouabain and related substances are cardiacglycosides derived from plants. The main pharmacodynamic property ofcardiac glycosides is the ability to increase the force of myocardialcontraction in a dose-dependent manner (positive inotropic effect). Themost probable explanation for the direct positive inotropic effect isthe ability of cardiac glycosides to inhibit membrane-bound(Na⁺+K⁺)-activated adenosine triphosphatase [(Na⁺+K⁺)-ATPase] (Hoffman,B. F. and J. T. Bigger, Jr., “Digitalis and Allied Cardiac Glycosides”in The Pharmacological Basis of Therapeutics, eds. Goodman and Gilman,p. 732, (1980)). The hydrolysis of adenosine triphosphate (ATP) by thisenzyme provides the energy for the sodium potassium pump.

[0004] The precise structural region of (Na⁺+K⁺)-ATPase that regulatescardiac function is not well understood. Hence, relatively little isknown about the endogenous regulation of (Na⁺+K⁺)-ATPase. Catecholamines(Phillis, J. W., Cell, Tissue and Organ Cultures in Neurobiology, pp.93-97 (1978); Horwitz, B. A., Fed Proc., 38:2170-2176 (1979)), thyroidhormone (Smith, T. J. and I. S. Edelman, Fed. Proc., 38:2150-2153(1979)), aldosterone (Rossier, B. C., et al., Science, 12:483-487(1987)), linoleic and linolenic acids (Bidard, J. N., et al., Biochem.Biophys. Acta., 769:245 (1984); Tamura, M., et al., J. Biol. Chem.,260:9672 (1985); and vanadium (Cantley, L. C., Jr., et al., J. Biol.Chem., 243:7361-7368 (1978)) have all been linked to either direct orindirect effects on enzyme activity.

[0005] Because of their positive inotropic effect, cardiac glycosides(e.g., digitalis and ouabain) are unrivaled in value for the treatmentof heart failure. Cardiac glycosides are most frequently usedtherapeutically to increase the adequacy of the circulation in patientswith congestive heart failure and to slow the ventricular rate in thepresence of atrial fibrillation and flutter.

[0006] However, cardiac glycosides have narrow therapeutic indices andtheir use is frequently accompanied by toxic effects that can be severeor lethal. The most important toxic effects, in terms of risk to thepatient, are those that involve the heart (e.g., abnormalities ofcardiac rhythm and disturbances of atrio-ventricular conduction).Gastrointestinal disorders, neurological effects, anorexia, blurredvision, nausea and vomiting are other common cardiac glycoside-inducedreactions. Consequently, there is a need in the art for positiveinotropic agents which overcome the disadvantages associated with knownagents, as well as a need for further information on the mechanisms andreceptors associated with cardiac muscle contractility.

[0007] It would be highly beneficial to provide patients with atherapeutic composition wherein the cardiac regulatory functions of(Na⁺+K⁺)-ATPase are specifically regulated. Moreover, the identificationof the key structural regions and amino acids of the (Na⁺+K⁺)-ATPasewould be of great importance in developing more specific therapeuticmolecules, which specifically regulate the cardiac function and differin characteristics from currently available digitalis glycosides.

SUMMARY OF THE INVENTION

[0008] The present invention provides for the identification of the keyfunctional sites of (Na⁺+K⁺)-ATPase and also of inotropic agents whichdirectly participate in the regulation of cardiac contraction. Inparticular, the inotropic agents are antibodies which bind to thesefunctional sites (epitopes) in the α-subunit of (Na⁺+K⁺)-ATPase andinduce a positive inotropic effect.

[0009] In particular, the invention provides for isolated and/orpurified antibodies (including both exogenous and endogenous) which bindto the structural binding site of (Na⁺+K⁺)-ATPase and regulate(Na⁺+K⁺)-ATPase functions. This is of importance in the treatment ofheart disease and other muscle contraction diseases. The antibodies ofthe invention are polyclonal and/or antisera, monoclonal, and/orhumanized antibodies.

[0010] The invention also provides for isolated and/or purifiedantibodies (including both exogenous and endogenous) which specificallyrecognize the amino acid sequences comprising RSATEEEPPNDD and/orDVEDSYGQQWTYEQR of the α-subunit of (Na⁺+K⁺)-ATPase enzyme. The bindingof the antibodies to these amino acid sequences of the α-subunit of(Na⁺+K⁺)-ATPase increase cardiac contraction and myocyte intracellulardiastolic and systolic calcium.

[0011] Preferred antibodies of the invention exert a positive inotropiceffect in cardiac myocytes, when they bind to their specific epitopes inthe α-subunit of (Na⁺+K⁺)-ATPase. The antibodies can be from antisera,polyclonal, monoclonal, and/or humanized antibodies. In a preferredembodiment the antibodies of the invention are used as a therapeuticagent to treat patients suffering from or susceptible to heart diseaseand/or other muscle contraction disorders.

[0012] In particular, the invention provides for purified peptides whichare used to generate inotropic antibodies when administered in vivo to apatient, suffering from or susceptible to heart disease and/or musclecontractile disorders. These peptides can be administered individuallyor in combination in a pharmaceutically acceptable carrier to a patient.

[0013] The invention also provides for isolated and/or purified peptidescomprising the amino acid sequence RSATEEEPPNDD and DVEDSYGQQWTYEQR orderivatives thereof (including isoforms), which are used to generateinotropic antibodies when administered in vivo to a patient sufferingfrom or susceptible to heart disease and/or myocyte contractiledisorders. These peptides can be administered individually or incombination in a pharmaceutically acceptable carrier to a patient.Preferred isoforms of such peptides (i.e. comprising the sequenceRSATEEEPPNDD or DVEDSYGQQWTYEQR) will also will generate such antibodiesand preferably will comprise an amino acid sequence that has only 1, 2,3, 4, 5, 6, 7 or 8 total amino acid differences from the sequence ofRSATEEEPPNDD or DVEDSYGQQWTYEQR, more preferably will comprise an aminoacid sequence that has only 1, 2, 3, or 4 total amino acid differencesfrom the sequence of RSATEEEPPNDD or DVEDSYGQQWTYEQR.

[0014] In accordance with the invention the peptides can be administeredin concentrations in a ratio of 1:1 or in varying ratios to each otheras defined by their concentration.

[0015] In another preferred embodiment, the invention provides forvectors which encode amino acid sequences which are used to generateinotropic antibodies when administered in vivo to a patient sufferingfrom or susceptible to heart disease and/or myocyte contractiledisorders. Preferably these vectors are under the control of tissuespecific promoters, in particular, cardiac tissue specific. Thesevectors are also preferably used in generating sera comprising inotropicantibodies using standard methods such as immunizing mammals.

[0016] In another preferred embodiment, the invention provides forvectors which encode the amino acid sequences RSATEEEPPNDD,DVEDSYGQQWTYEQR or isoforms (derivatives) thereof, are used to generateinotropic antibodies when administered in vivo to a patient sufferingfrom or susceptible to heart disease and/or myocyte contractiledisorders. Preferably these vectors are under the control of tissuespecific promoters, in particular, cardiac tissue specific. Thesepeptides are administered as a vaccine to a patient in need of suchtherapy, in order to generate endogenous inotropic antibodies.

[0017] The amino acids which are encoded by the vector stimulate theimmune system to generate antibodies which bind to their epitopes in theα-subunit of (Na⁺+K⁺)-ATPase, resulting in increased myocyteintracellular diastolic and systolic calcium. These antibodies, exert apositive inotropic effect in cardiac.

[0018] In a preferred embodiment, the invention provides for thetherapeutic use of antisera, polyclonal and monoclonal antibodies and/orhumanized antibodies that specifically bind to amino acid sequences of(Na⁺+K⁺)-ATPase enzyme and modulate the activity of the enzyme, fortreating patients suffering from or susceptible to heart disease and/ormuscle contractile disorders. These antibodies are also used to blockother molecules from binding to drug-interaction sites so that a patientsuffering from heart disorders such as, for example, arhythmia,tachyrhithmia and the like, are useful in regulating cardiaccontraction. The antibodies in this case would also function toeliminate of certain precipitating drugs, including negative inotropicagents (e.g., certain calcium channel blockers and antiarrhythmic drugslike disopyramide), cardiotoxins (e.g., amphetamines) and plasma volumeexpanders (e.g., nonsteroidal antiinflammatory agents andglucocorticoids).

[0019] In another preferred embodiment, the invention provides for amethod of generating antibodies, wherein binding of the antibodies to anepitope of the α-subunit of (Na⁺+K⁺)-ATPase exerts a positive inotropiceffect in cardiac myocytes, comprising:

[0020] generating amino acid sequences corresponding to overlappingpeptide fragments, and variants thereof, of the α-subunit of(Na⁺+K⁺)-ATPase (including the α-subunit of one or more isoforms of(Na⁺+K⁺)-ATPase); and,

[0021] obtaining antibodies specific for each peptide fragment bystandard methods; and,

[0022] determining the effects of the antibodies on intracellulardiastolic and systolic calcium levels and cell shortenings as comparedto controls.

[0023] The antibodies produced by this method, when they bind to theirantigenic sites in the α-subunit of (Na⁺+K⁺)-ATPase (including theα-subunit of one or more isoforms of (Na⁺+K⁺)-ATPase) exert a positiveinotropic effect in cardiac myocytes. The antibodies can be fromantisera, polyclonal antibodies, monoclonal antibodies, and/or humanizedantibodies. These antibodies are also used in immunoassays (e.g. RIA,ELISA, etc.) for diagnosing different heart and contractile disorders.

[0024] In another preferred embodiment, the invention provides for amethod for diagnosing heart failure and/or contractile disorderscomprising:

[0025] isolating heart tissue using standard methods; and,

[0026] obtaining cell cultures from the heart tissue using standardmethods; and,

[0027] allowing the binding of inotropic antibodies to specificepitopes; and,

[0028] measuring intracellular diastolic and systolic calcium and cellshortenings.

[0029] Preferably, the molecules of the invention are administered to apatient in an effective therapeutic amount to treat the patientsuffering from or susceptible to heart disease and/or muscle contractiledisorders.

[0030] In another preferred embodiment, the antibodies are administeredto a patient (e.g. as a vaccine-type agent or inotrpoic reagent) in atherapeutically effective amount to block other molecules from bindingto drug-interaction sites of (Na⁺+K⁺)-ATPase, wherein the patient issuffering from or susceptible to arhythmias, tachyrhithmias and thelike.

[0031] The invention also provides for identifying molecules whichtarget and block the RSATEEEPPNDD and/or DVEDSYGQQWTYEQR (orisoforms/derivatives thereof) site of α-subunit of the (Na⁺+K⁺)-ATPase(including the α-subunit of one or more isoforms of (Na⁺+K⁺)-ATPase),comprising:

[0032] contacting a myocyte with a desired molecule; and,

[0033] measuring the intracellular diastolic and systolic Ca²⁺; and,

[0034] measuring cell shortening and heart function; whereby,

[0035] identifying molecules useful for therapy of patients sufferingfrom or susceptible to heart disease and other contractile disorders.

[0036] Such molecules are used to generate inotropic antibodies and/orgenerate peptide-based vaccines as therapeutic agents in patientssuffering from and/or susceptible to heart disease and other contractiledisorders.

[0037] The Jianye-2 peptide (comprising or consisting of sequenceRSATEEEPPNDD) as disclosed herein is a particularly preferred peptide.Isoforms (e.g. differing in sequence by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10amino acids, preferably 1, 2, 3, 4, 5, 6, 7 or 8 amino acid differences,more preferably 1, 2, 3, or 4 amino acid differences) of the Jianye-2peptide also are preferred and those amino acid differences may reflectdifferences among species.

[0038] The KX-1 peptide (comprising or consisting of sequenceDVEDSYGQQWTYEQR) as disclosed herein is a further particularly preferredpeptide. Isoforms (e.g. differing in sequence by 1, 2, 3, 4, 5, 6, 7, 8,9 or 10 amino acids, preferably 1, 2, 3, 4, 5, 6, 7 or 8 amino aciddifferences, more preferably 1, 2, 3, or 4 amino acid differences) ofthe KX-1 peptide also are preferred and those amino acid differences mayreflect differences among species.

[0039] Also preferred are antibodies and vaccines made against a regionof the H1-H2 domain of the (Na⁺+K⁺)-ATPase (such as the Jianye-2 peptideor isoform thereof).

[0040] Additionally preferred are antibodies and vaccines made against aregion of the H7-H8 domain of the (Na⁺+K⁺)-ATPase (such as the KX-1peptide or isoform thereof).

[0041] Other aspects of the invention are described infra.

[0042] Definitions

[0043] As used herein, “inotropic agents” or “inotropic antibodies” willbe used interchangeably and refers to the effect such agents produce,i.e. improves cardiac output by increasing the force of myocardialmuscle contraction. “Positive inotropic effect” means that thecontractility of the cells is enhanced in a dose-dependent manner. Apositive inotropic effect-producing amount of antibodies or peptides ofthe invention can be administered to a “mammalian host” (e.g., a human)to treat cardiac malfunction (e.g., congestive heart failure, paroxysmalatrial tachycardia, atrial fibrillation and flutter). Administration canbe either enteral (i.e., oral) or parenteral (e.g., via intravenous,subcutaneous or intramuscular injection).

[0044] As used herein, a “vector” (sometimes referred to as genedelivery or gene transfer “vehicle”) refers to a macromolecule orcomplex of molecules comprising a polynucleotide to be delivered to ahost cell, either in vitro or in vivo. The polynucleotide to bedelivered may comprise a coding sequence of interest in gene therapy.Vectors include, for example, viral vectors (such as adenoviruses(“Ad”), adeno-associated viruses (AAV), and retroviruses), liposomes andother lipid-containing complexes, and other macromolecular complexescapable of mediating delivery of a polynucleotide to a host cell. Theinvention also provides for vectors which are used for treating apatient suffering from or susceptible heart disease. Vectors can alsocomprise other components or functionalities that further modulate genedelivery and/or gene expression, or that otherwise provide beneficialproperties to the targeted cells. As described and illustrated in moredetail below, such other components include, for example, componentsthat influence binding or targeting to cells (including components thatmediate cell-type or tissue-specific binding); components that influenceuptake of the vector nucleic acid by the cell; components that influencelocalization of the polynucleotide within the cell after uptake (such asagents mediating nuclear localization); and components that influenceexpression of the polynucleotide. Such components also might includemarkers, such as detectable and/or selectable markers that can be usedto detect or select for cells that have taken up and are expressing thenucleic acid delivered by the vector. Such components can be provided asa natural feature of the vector (such as the use of certain viralvectors which have components or functionalities mediating binding anduptake), or vectors can be modified to provide such functionalities. Alarge variety of such vectors are known in the art and are generallyavailable.

[0045] As used herein, the term “administering a molecule to a cell”(e.g., an expression vector, nucleic acid, peptide, a delivery vehicle,agent, and the like) refers to transducing, transfecting,microinjecting, electroporating, or shooting, the cell with themolecule. In some aspects, molecules are introduced into a target cellby contacting the target cell with a delivery cell (e.g., by cell fusionor by lysing the delivery cell when it is in proximity to the targetcell).

[0046] A cell has been “transformed”, “transduced”, or “transfected” byexogenous or heterologous nucleic acids when such nucleic acids havebeen introduced inside the cell. Transforming DNA may or may not beintegrated (covalently linked) with chromosomal DNA making up the genomeof the cell. In prokaryotes, yeast, and mammalian cells for example, thetransforming DNA may be maintained on an episomal element, such as aplasmid. In a eukaryotic cell, a stably transformed cell is one in whichthe transforming DNA has become integrated into a chromosome so that itis inherited by daughter cells through chromosome replication. Thisstability is demonstrated by the ability of the eukaryotic cell toestablish cell lines or clones comprised of a population of daughtercells containing the transforming DNA. A “clone” is a population ofcells derived from a single cell or common ancestor by mitosis. A “cellline” is a clone of a primary cell that is capable of stable growth invitro for many generations (e.g., at least about 10).

[0047] As used herein, “molecule” is used generically to encompass anyvector, antibody, protein, drug and the like which are used in therapyand can be detected in a patient by the methods of the invention. Forexample, multiple different types of nucleic acid delivery vectorsencoding different types of genes which may act together to promote atherapeutic effect, or to increase the efficacy or selectivity of genetransfer and/or gene expression in a cell. The nucleic acid deliveryvector may be provided as naked nucleic acids or in a delivery vehicleassociated with one or more molecules for facilitating entry of anucleic acid into a cell. Suitable delivery vehicles include, but arenot limited to: liposomal formulations, polypeptides; polysaccharides;lipopolysaccharides, viral formulations (e.g., including viruses, viralparticles, artificial viral envelopes and the like), cell deliveryvehicles, and the like.

[0048] A “recombinant viral vector” refers to a viral vector comprisingone or more heterologous genes or sequences. Since many viral vectorsexhibit size-constraints associated with packaging, the heterologousgenes or sequences are typically introduced by replacing one or moreportions of the viral genome. Such viruses may becomereplication-defective, requiring the deleted function(s) to be providedin trans during viral replication and encapsidation (by using, e.g., ahelper virus or a packaging cell line carrying genes necessary forreplication and/or encapsidation) (see, e.g., the references andillustrations below). Modified viral vectors in which a polynucleotideto be delivered is carried on the outside of the viral particle havealso been described (see, e.g., Curiel, D T, et al. PNAS 88: 8850-8854,1991).

[0049] Viral “packaging” as used herein refers to a series ofintracellular events that results in the synthesis and assembly of aviral vector. Packaging typically involves the replication of the“pro-viral genome”, or a recombinant pro-vector typically referred to asa “vector plasmid” (which is a recombinant polynucleotide than can bepackaged in an manner analogous to a viral genome, typically as a resultof being flanked by appropriate viral “packaging sequences”), followedby encapsidation or other coating of the nucleic acid. Thus, when asuitable vector plasmid is introduced into a packaging cell line underappropriate conditions, it can be replicated and assembled into a viralparticle. Viral “rep” and “cap” genes, found in many viral genomes, aregenes encoding replication and encapsidation proteins, respectively. A“replication-defective” or “replication-incompetent” viral vector refersto a viral vector in which one or more functions necessary forreplication and/or packaging are missing or altered, rendering the viralvector incapable of initiating viral replication following uptake by ahost cell. To produce stocks of such replication-defective viralvectors, the virus or pro-viral nucleic acid can be introduced into a“packaging cell line” that has been modified to contain genes encodingthe missing functions which can be supplied in trans). For example, suchpackaging genes can be stably integrated into a replicon of thepackaging cell line or they can be introduced by transfection with a“packaging plasmid” or helper virus carrying genes encoding the missingfunctions.

[0050] A “detectable marker gene” is a gene that allows cells carryingthe gene to be specifically detected (e.g., distinguished from cellswhich do not carry the marker gene). A large variety of such markergenes are known in the art. Preferred examples thereof includedetectable marker genes which encode proteins appearing on cellularsurfaces, thereby facilitating simplified and rapid detection and/orcellular sorting. By way of illustration, the lacZ gene encodingbeta-galactosidase can be used as a detectable marker, allowing cellstransduced with a vector carrying the lacZ gene to be detected bystaining, as described below.

[0051] A “selectable marker gene” is a gene that allows cells carryingthe gene to be specifically selected for or against, in the presence ofa corresponding selective agent. By way of illustration, an antibioticresistance gene can be used as a positive selectable marker gene thatallows a host cell to be positively selected for in the presence of thecorresponding antibiotic. Selectable markers can be positive, negativeor bifunctional. Positive selectable markers allow selection for cellscarrying the marker, whereas negative selectable markers allow cellscarrying the marker to be selectively eliminated. A variety of suchmarker genes have been described, including bifunctional (i.e.positive/negative) markers (see, e.g., WO 92/08796, published May 29,1992, and WO 94/28143, published Dec. 8, 1994). Such marker genes canprovide an added measure of control that can be advantageous in genetherapy contexts. “Treatment” or “therapy” as used herein also refers toadministering, to an individual patient, agents that are capable ofeliciting a prophylactic, curative or other beneficial effect in theindividual.

[0052] “Gene therapy” as used herein refers to administering, to anindividual patient, vectors comprising a therapeutic gene.

[0053] A “therapeutic polynucleotide” or “therapeutic gene” refers to anucleotide sequence that is capable, when transferred to an individual,of eliciting a prophylactic, curative or other beneficial effect in theindividual.

[0054] The term “treatment” or grammatical equivalents encompasses theimprovement and/or reversal of the symptoms of heart failure (i.e, theability of the heart to pump blood). “Improvement in the physiologicfunction” of the heart can be assessed using any of the measurementsdescribed herein (e.g., measurement of ejection fraction, fractionalshortening, left ventricular internal dimension, heart rate, etc. inresponse to isoproterenol and/or norepinephrine), as well as any effectupon the patient's survival. A compound which causes an improvement inany parameter associated with heart failure when used in the screeningmethods of the instant invention may thereby be identified as atherapeutic compound.

[0055] The term “individual” as used herein refers to vertebrates,particularly members of the mammalian species and includes but is notlimited to, domestic animals, sports animals, primates and humans; moreparticularly, the term refers to humans.

[0056] As used herein, the term “heart failure” is broadly used to meanany condition that reduces the ability of the heart to pump blood. As aresult, congestion and edema develop in the tissues. Most frequently,heart failure is caused by decreased contractility of the myocardium,resulting from reduced coronary blood flow; however, many other factorsmay result in heart failure, including damage to the heart valves,vitamin deficiency, and primary cardiac muscle disease. Though theprecise physiological mechanisms of heart failure are not entirelyunderstood, heart failure is generally believed to involve disorders inseveral cardiac autonomic properties, including sympathetic,parasympathetic, and baroreceptor responses. The phrase “manifestationsof heart failure” is used broadly to encompass all of the sequelaeassociated with heart failure, such as shortness of breath, pittingedema, an enlarged tender liver, engorged neck veins, pulmonary ralesand the like including laboratory findings associated with heartfailure.

[0057] As used herein, “contractile disorders” refers to the abnormalcontractile response of muscle cells as compared to normal muscle cells.Examples of such disorders are arhythmia, tachyrhithmia, and the like.

[0058] A “polynucleotide” refers to a polymeric form of nucleotides ofany length, either ribonucleotides or deoxyribonucleotides, or analogsthereof. This term refers to the primary structure of the molecule, andthus includes double- and single-stranded DNA, as well as double- andsingle-stranded RNA. It also includes modified polynucleotides such asmethylated and/or capped polynucleotides.

[0059] “Recombinant,” as applied to a polynucleotide, means that thepolynucleotide is the product of various combinations of cloning,restriction and/or ligation steps, and other procedures that result in aconstruct that is distinct from a polynucleotide found in nature.

[0060] A “gene” refers to a polynucleotide or portion of apolynucleotide comprising a sequence that encodes a protein. For mostsituations, it is desirable for the gene to also comprise a promoteroperably linked to the coding sequence in order to effectively promotetranscription. Enhancers, repressors and other regulatory sequences mayalso be included in order to modulate activity of the gene, as is wellknown in the art. (See, e.g., the references cited below).

[0061] The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably to refer to polymers of amino acids of any length. Theseterms also include proteins that are post-translationally modifiedthrough reactions that include glycosylation, acetylation andphosphorylation.

[0062] The terms “variant”, “derivative” and “amino acid sequencevariant” are used interchangeably and designate polypeptides in whichone or more amino acids are added and/or substituted and/or deletedand/or inserted at the N- or C-terminus or anywhere within thecorresponding native sequence. In various embodiments, a “variant”polypeptide usually has at least about 75% amino acid sequence identity,or at least about 80% amino acid sequence identity, preferably at leastabout 85% amino acid sequence identity, even more preferably at leastabout 90% amino acid sequence identity, and most preferably at leastabout 95% amino acid sequence identity with the amino acid sequence ofthe corresponding native sequence polypeptide.

[0063] An “effective amount” is an amount sufficient to effectbeneficial or desired clinical results. An effective amount can beadministered in one or more administrations. The antibodies, peptides orvectors used as vaccines of the present invention can be administered toa patient at therapeutically effective doses to treat (includingprevention) heart disease and/or other muscular contractile disorders. Atherapeutically effective dose refers to that amount of the compoundsufficient to result in desired treatment.

[0064] As used herein, the term “fragment or segment”, as applied to apolypeptide, will ordinarily be at least about 5 contiguous amino acids,typically at least about 10 contiguous amino acids, more typically atleast about 20 contiguous amino acids, usually at least about 30contiguous amino acids, preferably at least about 40 contiguous aminoacids, more preferably at least about 50 contiguous amino acids, andeven more preferably at least about 60 to 80 or more contiguous aminoacids in length. “Overlapping fragments” as used herein, refer tocontiguous peptide fragments which begin at the amino terminal end of aprotein and end at the carboxy terminal end of the protein. Each peptidefragment has at least about one contiguous amino acid position in commonwith the next peptide fragment, more preferably at least about threecontiguous amino acid positions in common, most preferably at leastabout ten contiguous amino acid positions in common.

[0065] As used herein, the term “substantially pure” describes acompound (e.g., a protein or polypeptide) which has been separated fromcomponents which naturally accompany it. Typically, a compound issubstantially pure when at least 10%, more preferably at least 20%, morepreferably at least 50%, more preferably at least 60%, more preferablyat least 75%, more preferably at least 90%, and even more preferably atleast 99%, of the total material (by volume, by wet or dry weight, or bymole percent or mole fraction) in a sample is the compound of interest.Purity can be measured by any appropriate method. In the case ofpolypeptides, for example, purity can be measured by columnchromatography, polyacrylamide gel electrophoresis, or HPLC analysis. Acompound such as a protein is also substantially purified when it isessentially free of naturally associated components or when it isseparated from the native contaminants which accompany it in its naturalstate.

[0066] A “heterologous” component refers to a component that isintroduced into or produced within a different entity from that in whichit is naturally located. For example, a polynucleotide derived from oneorganism and introduced by genetic engineering techniques into adifferent organism is a heterologous polynucleotide which, if expressed,can encode a heterologous polypeptide. Similarly, a promoter or enhancerthat is removed from its native coding sequence and operably linked to adifferent coding sequence is a heterologous promoter or enhancer.

[0067] A “substantially pure nucleic acid”, as used herein, refers to anucleic acid sequence, segment, or fragment which has been purified fromthe sequences which flank it in a naturally occurring state, e.g., a DNAfragment which has been removed from the sequences which are normallyadjacent to the fragment such as the sequences adjacent to the fragmentin a genome in which it naturally occurs. The term also applies tonucleic acids which have been substantially purified from othercomponents which naturally accompany the nucleic acid, e.g., RNA or DNA,which has been purified from proteins which naturally accompany it inthe cell.

[0068] “Homologous”, as used herein, refers to the subunit sequencesimilarity between two polymeric molecules, e.g., between two nucleicacid molecules such as two DNA molecules, or two polypeptide molecules.When a subunit position in both of the two molecules is occupied by thesame monomeric subunit (e.g., if a position in each of two DNA moleculesis occupied by adenine) then they are homologous at that position. Thehomology between two sequences is a direct function of the number ofmatching or homologous positions. For example, if 5 of 10 positions intwo compound sequences are matched or homologous then the two sequencesare 50% homologous, if 9 of 10 are matched or homologous, the twosequences share 90% homology. By way of example, the DNA sequences 3′ATTGCC 5′ and 3′ TTTCCG 5′ share 50% homology.

[0069] A “promoter,” as used herein, refers to a polynucleotide sequencethat controls transcription of a gene or coding sequence to which it isoperably linked. A large number of promoters, including constitutive,inducible and repressible promoters, from a variety of differentsources, are well known in the art and are available as or within clonedpolynucleotide sequences (from, e.g., depositories such as the ATCC aswell as other commercial or individual sources).

[0070] An “enhancer,” as used herein, refers to a polynucleotidesequence that enhances transcription of a gene or coding sequence towhich it is operably linked. A large number of enhancers, from a varietyof different sources are well known in the art and available as orwithin cloned polynucleotide sequences (from, e.g., depositories such asthe ATCC as well as other commercial or individual sources). A number ofpolynucleotides comprising promoter sequences (such as the commonly-usedCMV promoter) also comprise enhancer sequences. “Operably linked” refersto a juxtaposition, wherein the components so described are in arelationship permitting them to function in their intended manner. Apromoter is operably linked to a coding sequence if the promotercontrols transcription of the coding sequence. Although an operablylinked promoter is generally located upstream of the coding sequence, itis not necessarily contiguous with it. An enhancer is operably linked toa coding sequence if the enhancer increases transcription of the codingsequence. Operably linked enhancers can be located upstream, within ordownstream of coding sequences. A polyadenylation sequence is operablylinked to a coding sequence if it is located at the downstream end ofthe coding sequence such that transcription proceeds through the codingsequence into the polyadenylation sequence.

[0071] A “replicon” refers to a polynucleotide comprising an origin ofreplication which allows for replication of the polynucleotide in anappropriate host cell. Examples include replicons of a target cell intowhich a heterologous nucleic acid might be integrated (e.g., nuclear andmitochondrial chromosomes), as well as extrachromosomal replicons (suchas replicating plasmids and episomes).

[0072] As used herein, the term “antibody” refers to a polypeptide orgroup of polypeptides which are comprised of at least one bindingdomain, where an antibody binding domain is formed from the folding ofvariable domains of an antibody molecule to form three-dimensionalbinding spaces with an internal surface shape and charge distributioncomplementary to the features of an antigenic determinant of an antigen,which allows an immunological reaction with the antigen. Antibodiesinclude recombinant proteins comprising the binding domains, as wells asfragments, including Fab, Fab′, F(ab)₂, and F(ab′)₂ fragments.

[0073] The term “polyclonal” refers to antibodies that are heterogeneouspopulations of antibody molecules derived from the sera of animalsimmunized with an antigen or an antigenic functional derivative thereof.For the production of polyclonal antibodies, various host animals may beimmunized by injection with the antigen. Various adjuvants may be usedto increase the immunological response, depending on the host species.

[0074] “Monoclonal antibodies” are substantially homogenous populationsof antibodies to a particular antigen. They may be obtained by anytechnique which provides for the production of antibody molecules bycontinuous cell lines in culture. Monoclonal antibodies may be obtainedby methods known to those skilled in the art. See, for example, Kohler,et al., Nature 256:495-497, 1975, and U.S. Pat. No. 4,376,110.

[0075] As used herein, an “antigenic determinant” is the portion of anantigen molecule that determines the specificity of the antigen-antibodyreaction. An “epitope” refers to an antigenic determinant of apolypeptide. An epitope can comprise as few as 3 amino acids in aspatial conformation which is unique to the epitope. Generally anepitope consists of at least 6 such amino acids, and more usually atleast 8-10 such amino acids. Methods for determining the amino acidswhich make up an epitope include x-ray crystallography, 2-dimensionalnuclear magnetic resonance, and epitope mapping e.g. the Pepscan methoddescribed by H. Mario Geysen et al. 1984. Proc. Natl. Acad. Sci. U.S.A.81:3998-4002; PCT Publication No. WO 84/03564; and PCT Publication No.WO 84/03506.

[0076] The phrase “specifically (or selectively) binds” to an antibodyor “specifically (or selectively) immunoreactive with,” when referringto a protein or peptide, refers to a binding reaction that isdeterminative of the presence of the protein in a heterogeneouspopulation of proteins and other biologics. Thus, under designatedimmunoassay conditions, the specified antibodies bind to a particularprotein at least two times the background and do not substantially bindin a significant amount to other proteins present in the sample.Specific binding to an antibody under such conditions may require anantibody that is selected for its specificity for a particular protein.For example, polyclonal antibodies raised to marker “X” from specificspecies such as rat, mouse, or human can be selected to obtain onlythose polyclonal antibodies that are specifically immunoreactive withmarker “X” and not with other proteins, except for polymorphic variantsand alleles of marker “X”. This selection may be achieved by subtractingout antibodies that cross-react with marker “X” molecules from otherspecies. A variety of immunoassay formats may be used to selectantibodies specifically immunoreactive with a particular protein. Forexample, solid-phase ELISA immunoassays are routinely used to selectantibodies specifically immunoreactive with a protein (see, e.g., Harlow& Lane, Antibodies, A Laboratory Manual (1988), for a description ofimmunoassay formats and conditions that can be used to determinespecific immunoreactivity). Typically a specific or selective reactionwill be at least twice background signal or noise and more typicallymore than 10 to 100 times background.

[0077] Immunoassay” is an assay that uses an antibody to specificallybind an antigen (e.g., a marker). The immunoassay is characterized bythe use of specific binding properties of a particular antibody toisolate, target, and/or quantify the antigen.

BRIEF DESCRIPTION OF FIGURES

[0078]FIG. 1 (which includes FIGS. 1A through 1F) are photographsshowing the immunofluorescent staining of Jianye-2 antibody in ratcardiac myocytes and African green monkey CV-1 cells. FIGS. 1A-1B arephotographs of the confocal image of rat cardiac myocytes in thepresence of Jianye-2 (FIG. 1A) or with both Jianye-2 antibody andpeptide blocker (FIG. 1B). FIG. 1C shows the immunofluorescent stainingsof Jianye-2 antibody in a group of CV-1 cells at a magnification of400×. FIG. 1D is a photograph of a single CV-1 cell image at 3000×.FIGS. 1E-1F are photographs showing Jianye-2 antibody staining in thepresence of either 1 mM ouabain (FIG. 1E) or strophanthidin (FIG. 1F).The results indicate that Jianye-2 antibody binds to its antigenic siteof the (Na⁺+K⁺)-ATPase on the surface of the cell membrane.

[0079]FIG. 2 presents electrocardiograms showing results of the timecourse of rat heart cell contraction with or without Jianye-2 antibody.Time runs from left to right. Column A, shows the baselines of rat heartcell contraction. Columns B, C, D show the results obtained 10, 20, 30min after administration of either buffer (upper panel) or Jianye-2antibody (lower panel). Enzyme activity was monitored in cellhomogenates under the same experimental condition. The results show thatJianye-2 antibody enhanced rat heart cell contraction without inhibiting(Na⁺+K⁺)-ATPase activity.

[0080]FIG. 3 shows that Jianye-2 antibody markedly increasedintracellular Ca²⁺ contraction and demonstrates that intracellular Ca²⁺concentration is involved in the mechanisms of Jianye-2 antibodyenhanced heart cell contraction. The data represent a mean of 12independent experiments.

[0081]FIG. 4 is the dose-dependent contractile response of Jianye-2antibody in rat ventricular myocytes. The half maximal contractileresponse (EC50) is 35 nM. The data represent a mean of four independentexperiments.

[0082]FIG. 5 is a schematic representation of pressure-volume loops ofthe effect of Jianye-2 antibody on normal mouse heart left ventricle.Jianye-2 antibody (total 150 ml, 6.0 μM in PBS, pH 7.2) was administeredto the mouse heart at a rate of 5 ml/min. The results show that Jianye-2antibody dramatically induced a reversible positive inotropic effect asdemonstrated by the changes in ventricular pressure-volume loops withtime during the cardiac cycle. The results represent one of the sevensimilar experiments.

[0083]FIG. 6 shows the time- and concentration-dependent Jianye-2antibody effects on mouse heart contraction in vivo mouse model. Atdifferent fixed concentrations of Jianye-2 antibody as indicated in thefigure, the data are plotted in the form of percent of mouse heartcontraction as a function of time. Compared with the background control(at 0 min as 100%), Jianye-2 antibody increased mouse heart contractionsare expressed as percentages (%) for 5, 10, 15, and 20 min respectivelyafter administration of 3.4 μM (open circles) or 6.0 μM (black circles)of Jianye-2 antibody. Mouse heart contraction was 108(±5.3), 113(±7),116(±10), 122(±9.0) as shown in the open circles, and 115(±9.0),127(±21), 137(±16), and 143(±30) in black circles. First order rateconstants were 1.06 and 2.16%/min for 3.4 μM and 6.0 μM Jianye-2antibody, respectively. The data represent the mean of 5 independentdeterminations.

[0084]FIG. 7 shows that the KX-1 antibody enhanced the velocity ofshortening of rat ventricular myocyte and increased the force ofcontraction of the heart cells. Time runs from left to right. Column Arepresents the baselines of rat heart cell contraction. Columns B & C: 5and 16 minutes following administration of KX-1. The final concentrationof KX-1 was 0.74 μM. The results indicate that the KX-1 antibody is aninotropic agent.

[0085]FIG. 8 represents the changes of intracellular calcium transientfollowing the binding of KX-1 to the (Na⁺+K⁺)-ATPase and indicates thatKX-1 induced heart cell contraction is dependent on intracellular Ca²⁺increase.

[0086]FIG. 9 is a graph showing the effect of immunization with KX-1 onthe rat cardiac heart failure function. The peptide DVEDSYGQQWTYEQR wasinjected into the animal as a vaccine to reduce the rate of progressionof heart failure in the heart failure rat model. TiterMax Gold was usedas an adjuvant throughout the experiment. The results show thatendogenous KX-1 antibody generation significantly delayed the rate ofthe progression of heart failure in heart failure rats (red circles). Incontrast, cardiac function was significantly depressed in the controlheart failure rat without immunization with KX-1 antigen (blue circles).

DETAILED DESCRIPTION OF THE INVENTION

[0087] Antibody (Jianye-2 antibody), which recognizes the RSATEEEPPNDDpeptide (H1-H2 domain) of the α-subunit of the (Na⁺+K⁺)-ATPase, and KX-1antibody which recognizes the DVEDSYGQQWTYEQR peptide (H7-H8 domain) ofthe α-subunit of the (Na⁺+K⁺)-ATPase, have been found to increase thecontractility of ventricular myocytes which is important in thetreatment of muscle contractile disorders.

[0088] In a preferred embodiment, the invention provides for antisera,polyclonal and monoclonal antibodies and/or humanized antibodies thatspecifically bind to amino acid sequences of (Na⁺+K⁺)-ATPase, resultingin increased intracellular Ca²⁺ transients and contraction in intactmammalian heart cells and in living mouse heart.

[0089] In accordance with the invention, it is preferred that theantibodies specifically bind to peptides having an amino acid sequenceRSATEEEPPNDD (the antibody is referred to herein as the “Jianye-2”antibody), and DVEDSYGQQWTYEQR (the antibody referred to herein as the“KX-1” antibody), mutants or derivatives thereof. These peptides can beconjugated into polypeptides either directly or through a linker.However, the invention is not limited to these sequences but applies toany sequence in which antibodies can bind resulting in cardiac positiveinotropy. The Jianye-2 and KX-1 antibodies are described in detail inthe Examples which follow.

[0090] In a preferred embodiment, the invention provides for thetherapeutic use of antisera, polyclonal and monoclonal antibodies and/orhumanized antibodies that specifically bind to amino acid sequences of(Na⁺+K⁺)-ATPase enzyme and modulate the activity of the enzyme, fortreating patients suffering from or susceptible to heart disease and/ormuscle contractile disorders. These antibodies are also used to blockother molecules from binding to drug-interaction sites so that a patientsuffering from heart disorders such as, for example, arhythmia,tachyrhithmia and the like, are useful in regulating cardiaccontraction. The antibodies in this case would also function toeliminate of certain precipitating drugs, including negative inotropicagents (e.g., certain calcium channel blockers and antiarrhythmic drugslike disopyramide), cardiotoxins (e.g., amphetamines) and plasma volumeexpanders (e.g., nonsteroidal antiinflammatory agents andglucocorticoids).

[0091] In another preferred embodiment, antibodies that bind to specificsequences of (Na⁺+K⁺)-ATPase and can produce cardiac positive inotropyare administered to patients in need of such therapy.

[0092] In another embodiment, the molecules of the invention are used asdiagnostic agents for heart disease or other contractile disorders, bydetecting, in standard assays, such as ELISAs, RIAs and the like,peptides which are indicative of contractile disorders.

[0093] In another preferred embodiment, the invention provides forpharmaceutical compositions comprising peptides which are administeredto patients resulting in the generation of antibodies which recognizesuch peptides resulting in the in vivo generation of inotropicantibodies. Particularly preferred peptides include, but are not limitedto peptides with amino acid sequence RSATEEEPPNDD and/orDVEDSYGQQWTYEQR, mutants and variants thereof.

[0094] In another preferred embodiment, the invention provides for avaccine which codes for amino acids which generate inotropic antibodieswhen administered in vivo to a patient in need of such therapy ortreatment.

[0095] In accordance with the invention, the antibodies of the inventionare also used as diagnostic agents which detect muscle contractiledisorders, especially, for example, in the heart. In one embodiment, anyof the above-described molecules can be labeled, either detectably, aswith a radioisotope, a paramagnetic atom, a fluorescent moiety, anenzyme, etc. in order to facilitate its detection in, for example, insitu or in vivo assays. The molecules may be labeled with reagents suchas biotin, in order to, for example, facilitate their recovery, and/ordetection.

[0096] In another preferred embodiment, where the antibodies or theirfragments are intended for therapeutic purposes, it is desirable to“humanize” them in order to attenuate any immune reaction. Humanizedantibodies may be produced, for example by replacing an immunogenicportion of an antibody with a corresponding, but non-immunogenic portion(i.e. chimeric antibodies) (Robinson, R. R. et al., International PatentPublication PCT/U.S.86/02269; Akira, K. et al., European PatentApplication 184,187; Taniguchi, M., European Patent Application 171,496;Morrison, S. L. et al., European Patent Application 173,494; Neuberger,M. S. et al., PCT Application WO 86/01533; Cabilly, S. et al., EuropeanPatent Application 125,023; Better, M. et al., Science 240:1041-1043(1988); Liu, A. Y. et al. Proc. Natl. Acad. Sci. USA 84:3439-3443(1987); Liu, A. Y. et al., J. Immunol. 139:3521-3526 (1987); Sun, L. K.et al., Proc. Natl. Acad. Sci. USA 84:214-218 (1987); Nishimura, Y. etal., Canc. Res. 47:999-1005 (1987); Wood, C. R. et al., Nature314:446-449 (1985)); Shaw et al., J. Natl. Cancer Inst. 80:1553-1559(1988); all of which references are incorporated herein by reference).General reviews of “humanized” chimeric antibodies are provided byMorrison, S. L. (Science, 229:1202-1207 (1985)) and by Oi, V. T. et al.,BioTechniques 4:214 (1986); which references are incorporated herein byreference).

[0097] The present invention provides humanized antibody moleculesspecific for peptides having an amino acid sequence RSATEEEPPNDD,DVEDSYGQQWTYEQR or derivatives thereof. However, the invention is notlimited to these sequences but applies to any sequence in whichantibodies can bind resulting in cardiac positive inotropy. Inaccordance with the present invention, the humanized antibodiescomprised antigen specific regions in which at least parts of the CDRsof the heavy and/or light chain variable regions of a human antibody(the receptor antibody) have been substituted by analogous parts of CDRsof a murine monoclonal antibody and the humanized antibody canspecifically bind to the same antigen as, for example, the Jianye-2antibody. In a preferred embodiment of the subject invention, the CDRregions of the humanized Jianye-2 is derived from rabbits as describedin the examples which follow. Some of the humanized antibodies describedherein contain some alterations of the acceptor antibody, i.e., human,heavy and/or light chain variable domain framework regions that arenecessary for retaining binding specificity of the donor monoclonalantibody. In other words, the framework region of some embodiments thehumanized antibodies described herein does not necessarily consist ofthe precise amino acid sequence of the framework region of a naturaloccurring human antibody variable region, but contains varioussubstitutions that improve the binding properties of a humanizedantibody region that is specific for the same target as the Jianye-2 orKX-1 antibodies. A minimal number of substitutions are made to theframework region in order to avoid large-scale introductions ofnon-human framework residues and to ensure minimal immunogenicity of thehumanized antibody in humans. The donor monoclonal antibodies of thepresent invention Jianye-2 or KX-1 antibodies, which are specific forthe rat α-subunit of (Na⁺+K⁺)-ATPase i.e., RSATEEEPPNDD andDVEDSYGQQWTYEQR peptides respectively.

[0098] The humanized antibodies compositions of the invention or othertherapeutic agents of the invention may be administered to a patient ina variety of ways. Preferably, the pharmaceutical compositions may beadministered parenterally, i.e., subcutaneously, intramuscularly orintravenously. Thus, this invention provides compositions for parenteraladministration which comprise a solution of the human monoclonalantibody or a cocktail thereof dissolved in an acceptable carrier,preferably an aqueous carrier. A variety of aqueous carriers can beused, e.g., water, buffered water, 0.4% saline, 0.3% glycine and thelike. These solutions are sterile and generally free of particulatematter. These compositions may be sterilized by conventional, well knownsterilization techniques. The compositions may contain pharmaceuticallyacceptable auxiliary substances as required to approximate physiologicalconditions such as pH adjusting and buffering agents, toxicity adjustingagents and the like, for example sodium acetate, sodium chloride,potassium chloride, calcium chloride, sodium lactate, etc. Theconcentration of antibody in these formulations can vary widely, e.g.,from less than about 0.5%, usually at or at least about 1% to as much as15 or 20% by weight and will be selected primarily based on fluidvolumes, viscosities, etc., in accordance with the particular mode ofadministration selected.

[0099] Actual methods for preparing parenterally administrablecompositions and adjustments necessary for administration to subjectswill be known or apparent to those skilled in the art and are describedin more detail in, for example, Remington's Pharmaceutical Science, 15thEd., Mack Publishing Company, Easton, Pa. (1980), which is incorporatedherein by reference.

[0100] As used herein, the term “pharmaceutically acceptable carrier”encompasses any of the standard pharmaceutical carriers, such as aphosphate buffered saline solution, water, and emulsions, such as anoil/water or water/oil emulsion, and various types of wetting agents.The compositions also can include stabilizers and preservatives. Forexamples of carriers, stabilizers and adjuvants, see Martin Remington'sPharm. Sci., 15th Ed. (Mack Publ. Co., Easton (1975)).

[0101] Toxicity and therapeutic efficacy of such compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD₅₀ (the dose lethal to50% of the population) and the ED₅₀ (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD₅₀/ED₅₀. Compounds exhibiting large therapeutic indices are preferred.While compounds that exhibit toxic side effects can be used, care shouldbe taken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

[0102] Data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage can vary within this range depending upon-the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose can beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound, which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used toaccurately determine useful doses in humans. Levels in plasma can bemeasured, for example, by high performance liquid chromatography. Atypical daily dose for the therapeutic molecules of the invention (i.e.,antibodies, peptides, vectors encoding peptides) of the presentinvention might range from about 1 μg/kg to about 100 mg/kg of patientbody weight or more per day, depending on the factors mentioned above,preferably about 10 μg/kg/day to 10 mg/kg/day.

[0103] Pharmaceutical compositions for use in accordance with thepresent invention can be formulated in conventional manner using one ormore physiologically acceptable carriers or excipients. Thus, thecompounds and their physiologically acceptable salts and solvates can beformulated for administration by intra venous or oral, buccal,parenteral or rectal administration.

[0104] For oral administration, the pharmaceutical compositions can takethe form of, for example, tablets or capsules prepared by conventionalmeans with pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulfate). Thetablets can be coated by methods well known in the art. Liquidpreparations for oral administration can take the form of, for example,solutions, syrups or suspensions, or they can be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations can be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations can also contain buffer salts, flavoring,coloring and sweetening agents as appropriate. Preparations for oraladministration can be suitably formulated to give controlled release ofthe active compound. For buccal administration the compositions can takethe form of tablets or lozenges formulated in conventional manner.

[0105] The compounds can be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection can be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionscan take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and can contain formulatory agents such as suspending,stabilizing or dispersing agents. Alternatively, the active ingredientcan be in powder form for constitution with a suitable vehicle, e.g.,sterile pyrogen-free water, before use. The compounds can also beformulated in rectal compositions such as suppositories or retentionenemas, e.g., containing conventional suppository bases such as cocoabutter or other glycerides.

[0106] In addition to the formulations described previously, thecompounds can also be formulated as a depot preparation. Such longacting formulations can be administered by implantation (for example,subcutaneously or intramuscularly) or by intramuscular injection. Thus,for example, the compounds can be formulated with suitable polymeric orhydrophobic materials (for example as an emulsion in an acceptable oil)or ion exchange resins, or as sparingly soluble derivatives, forexample, as a sparingly soluble salt.

[0107] The compositions can, if desired, be presented in a pack ordispenser device that can contain one or more unit dosage formscontaining the active ingredient. The pack can for example comprisemetal or plastic foil, such as a blister pack. The pack or dispenserdevice can be accompanied by instructions for administration.

[0108] The treatment can be monitored by various ways, includingechography and electrocardiograms. “Electrocardiogram” refers to agraphic tracing of variations in electrical potential caused by theexcitation of the heart muscle which may be detected at the bodysurface. “Electrocardiogram” may be abbreviated as “ECG” or “EKG”. Thesignals may be detected by means of metal electrodes attached to theextremities and chest wall, and may then be amplified by a sensitivevoltmeter such as the electrocardiograph. The ECG waveforms aregenerally labeled alphabetically beginning with the P wave, whichrepresents atrial depolarization. Approximately 0.16 seconds after theonset of the P wave, the QRS waves generally appear as a result ofdepolarization of the ventricular muscle, which initiates contraction ofthe ventricles. Finally, the T wave results from repolarization of theventricles, which represents the onset of ventricular relaxation. Theduration of the “T” wave cycle time is that time in a heart cycle whenit is most vulnerable to fibrillation, a condition where the cardiacmuscle fiber contracts asynchronously. Electrocardiography is furtherdescribed in Harrison's Principles of Internal Medicine, Thirteenth Ed.,McGraw-Hill, Inc., Chapter 189, pp. 954-966 (1994), the disclosures ofwhich are hereby incorporated herein by reference, in their entirety.

[0109] Echocardiography is the preferred method of monitoring treatmentusing the molecules of the invention. “Echocardiography” (Echo) usessound waves to form a picture of the heart valves and heart muscle. TheEcho machine sends sound waves to a transducer (a sound sensitiveinstrument) that is placed on the patient's chest. The sound waves arereflected by the heart walls (muscle) and heart valves, back to thetransducer, which changes the sound into a picture. There is no specialpreparation for this test. Gel is applied on the patient's chest and atransducer is placed over the heart area. Heart structures are examinedby changing the direction of the transducer. The sound waves cause nodiscomfort. When the test is completed the gel is wiped off easily.Thus, an Echo detects the changes and provides information about heartchamber size, wall motion, valve movements, and structural changes inand around the heart.

[0110] The invention also provides for vectors which are used fortreating a patient suffering from or susceptible heart disease. Vectorscan also comprise other components or functionalities that furthermodulate gene delivery and/or gene expression, or that otherwise providebeneficial properties to the targeted cells. As described andillustrated in more detail below, such other components include, forexample, components that influence binding or targeting to cells(including components that mediate cell-type or tissue-specificbinding); components that influence uptake of the vector nucleic acid bythe cell; components that influence localization of the polynucleotidewithin the cell after uptake (such as agents mediating nuclearlocalization); and components that influence expression of thepolynucleotide. Such components also might include markers, such asdetectable and/or selectable markers that can be used to detect orselect for cells that have taken up and are expressing the nucleic aciddelivered by the vector. Such components can be provided as a naturalfeature of the vector (such as the use of certain viral vectors whichhave components or functionalities mediating binding and uptake), orvectors can be modified to provide such functionalities. A large varietyof such vectors are known in the art and are generally available.

[0111] The practice of the present invention can suitably employ, unlessotherwise indicated, conventional techniques of molecular biology andthe like, which are within the skill of the art. Such techniques areexplained fully in the literature. See e.g., Molecular Cloning: ALaboratory Manual, (J. Sambrook et al., Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y., 1989); Current Protocols in Molecular Biology(F. Ausubel et al. eds., 1987 and updated); Essential Molecular Biology(T. Brown ed., IRL Press 1991); Gene Expression Technology (Goeddel ed.,Academic Press 1991); Methods for Cloning and Analysis of EukaryoticGenes (A. Bothwell et al. eds., Bartlett Publ. 1990); Gene Transfer andExpression (M. Kriegler, Stockton Press 1990); Recombinant DNAMethodology (R. Wu et al. eds., Academic Press 1989); PCR: A PracticalApproach (M. McPherson et al., IRL Press at Oxford University Press1991); Cell Culture for Biochemists (R. Adams ed., Elsevier SciencePublishers 1990); Gene Transfer Vectors for Mammalian Cells (J. Miller &M. Calos eds., 1987); Mammalian Cell Biotechnology (M. Butler ed.,1991); Animal Cell Culture (J. Pollard et al. eds., Humana Press 1990);Culture of Animal Cells, 2nd Ed. (R. Freshney et al. eds., Alan R. Liss1987); Flow Cytometry and Sorting (M. Melamed et al. eds., Wiley-Liss1990); the series Methods in Enzymology (Academic Press, Inc.);Techniques in Immunocytochemistry, (G. Bullock & P. Petrusz eds.,Academic Press 1982, 1983, 1985, 1989); Handbook of ExperimentalImmunology, (D. Weir & C. Blackwell, eds.); Cellular and MolecularImmunology (A. Abbas et al., W. B. Saunders Co. 1991, 1994); CurrentProtocols in Immunology (J. Coligan et al. eds. 1991); the series AnnualReview of Immunology; the series Advances in Immunology; OligonucleotideSynthesis (M. Gait ed., 1984); and Animal Cell Culture (R. Freshney ed.,IRL Press 1987).

[0112] Preferred vectors for use in the present invention include viralvectors, lipid-based vectors and other vectors that are capable ofdelivering DNA to non-dividing cells in vivo. Presently preferred areviral vectors, particularly replication-defective viral vectors(including, for example replication-defective adenovirus vectors andadeno-associated virus (AAV) vectors. For ease of production and use inthe present invention, replication-defective adenovirus vectors arepresently most preferred.

[0113] “Gene delivery,” “gene transfer,” and the like as used herein,are terms referring to the introduction of an exogenous polynucleotide(sometimes referred to as a “transgenes”) into a host cell, irrespectiveof the method used for the introduction. Such methods include a varietyof well-known techniques such as vector-mediated gene transfer (by,e.g., viral infection/transfection, or various other protein-based orlipid-based gene delivery complexes) as well as techniques facilitatingthe delivery of “naked” polynucleotides (such as electroporation, “genegun” delivery and various other techniques used for the introduction ofpolynucleotides). The introduced polynucleotide may be stably ortransiently maintained in the host cell. Stable maintenance typicallyrequires that the introduced polynucleotide either contains an origin ofreplication compatible with the host cell or integrates into a repliconof the host cell such as an extrachromosomal replicon (e.g., a plasmid)or a nuclear or mitochondrial chromosome. A number of vectors are knownto be capable of mediating transfer of genes to mammalian cells, as isknown in the art and described herein. Targeted vectors include vectors(such as viruses, non-viral protein-based vectors and lipid-basedvectors) in which delivery results in transgene expression that isrelatively limited to particular host cells or host cell types. By wayof illustration, therapeutic molecules, for example, nucleic acidsequences encoding for the peptides of the invention, to be delivered toa patient can be operably linked to heterologous tissue-specificpromoters thereby restricting expression to cells in that particulartissue.

[0114] “In vivo” gene delivery, gene transfer, gene therapy and the likeas used herein, are terms referring to the introduction of a vectorcomprising an exogenous polynucleotide directly into the body of anorganism, such as a human or non-human mammal, whereby the exogenouspolynucleotide is introduced to a cell of such organism in vivo.

[0115] The presently preferred means of in vivo delivery, is byinjection of the vector into a blood vessel directly supplying themyocardium, preferably by injection into a coronary artery. Suchinjection is preferably achieved by catheter introduced substantially(typically at least about 1 cm) within the ostium of one or bothcoronary arteries or one or more saphenous veins or internal mammaryartery grafts or other conduits delivering blood to the myocardium.

[0116] By injecting the vector stock, preferably containing no wild-typevirus, deeply into the lumen of one or both coronary arteries (or graftsand other vascular conduits), preferably into both the right and leftcoronary arteries (or grafts and other vascular conduits), andpreferably in an amount of about 10⁷-10¹³ viral particles as determinedby optical densitometry (more preferably 10⁹-10¹¹ viral particles), itis possible to locally transfect a desired number of cells, especiallycardiac myocytes, with genes that encode proteins that regulate cardiaccontraction, such as, for example, the peptides discussed infra, therebymaximizing therapeutic efficacy of gene transfer, and minimizingundesirable effects at extracardiac sites and the possibility of aninflammatory response to viral proteins. Vector constructs that arespecifically targeted to the myocardium, such as vectors incorporatingmyocardial-specific binding or uptake components, and/or whichincorporate inotropic molecules, for example, the peptides describedabove, that are under the control of myocardial-specific transcriptionalregulatory sequences (e.g., ventricular myocyte-specific promoters) canbe used in place of or, preferably, in addition to such directedinjection techniques as a means of further restricting expression to themyocardium, especially the ventricular myocytes. For vectors that canelicit an immune response, it is preferable to inject the vectordirectly into a blood vessel supplying the myocardium as describedabove, although the additional techniques for restricting the potentialfor extracardiac expression can also be employed. Additional referencesdescribing cell types found in the blood vessels, and the structure ofthe vasculature which may be useful in the methods of the presentinvention include the following: W. Bloom & D. Fawcett, A Textbook ofHistology, 10th Ed., (W. B. Saunders Co. 1975). Methods of uses of genetransfer for the treatment or prevention of disease, including heartdisease are described, e.g., Methods in Molecular Biology, Vol. 7: GeneTransfer and Expression Protocols, Murray, E. (ed.), Humana Press,Clifton, N.J. (1991); Mazur et al., Molecular and Cellular Pharmacology,21:104-111, 1994; French, Herz 18:222-229, 1993; Williams, AmericanJournal of Medical Sciences 306:129-136, 1993; and Schneider and French,Circulation 88:1937-1942, 1993.

[0117] “Vasculature” or “vascular” are terms referring to the system ofvessels carrying blood (as well as lymph fluids) throughout themammalian body.

[0118] “Blood vessel” refers to any of the vessels of the mammalianvascular system, including arteries, arterioles, capillaries, venules,veins, sinuses, and vasa vasorum.

[0119] “Artery” refers to a blood vessel through which blood passes awayfrom the heart. Coronary arteries supply the tissues of the heartitself, while other arteries supply the remaining organs of the body.The general structure of an artery consists of a lumen surrounded by amulti-layered arterial wall.

[0120] The invention also provides for methods for identifying peptidesand antibodies which are positive inotropic agents. To prepare anantibody that specifically binds to a region of the Na⁺, K⁺-ATPase,purified peptides or their nucleic acid sequences representing thedifferent subunits of Na⁺, K⁺-ATPase can be used. Using the purifiedpeptides or their nucleic acid sequences representing the differentsubunits of Na⁺, K⁺-ATPase, antibodies that specifically bind to adesired peptide can be prepared using any suitable methods known in theart. See, e.g., Coligan, Current Protocols in Immunology (1991); Harlow& Lane, Antibodies: A Laboratory Manual (1988); Goding, MonoclonalAntibodies: Principles and Practice (2d ed. 1986); and Kohler &Milstein, Nature 256:495-497 (1975). Such techniques include, but arenot limited to, antibody preparation by selection of antibodies fromlibraries of recombinant antibodies in phage or similar vectors, as wellas preparation of polyclonal and monoclonal antibodies by immunizinganimals (see, e.g., Huse et al., Science 246:1275-1281 (1989); Ward etal., Nature 341:544-546 (1989)); humanized antibodies; production ofantibodies by any of the methods discussed above. After the antibody isprovided, the specificity of the antibody can be detected using any ofsuitable immunological binding assays known in the art (see, e.g., U.S.Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168). Useful assaysinclude, for example, an enzyme immune assay (EIA) such as enzyme-linkedimmunosorbent assay (ELISA), a radioimmune assay (RIA), a Western blotassay, or a slot blot assay. These methods are also described in, e.g.,Methods in Cell Biology: Antibodies in Cell Biology, volume 37 (Asai,ed. 1993); Basic and Clinical Immunology (Stites & Terr, eds., 7th ed.1991); and Harlow & Lane, supra.

[0121] To determine whether these identified antibodies are positiveinotropic agents, standard assays such as those described in theExamples which follow can be used. For example, measurement of cellcontraction assays; confocal Ca²⁺ imaging; Na⁺, K⁺-ATPase activityassays and the like.

[0122] In another embodiment peptides that induce production ofinotropic antibodies in vivo and in vitro are preferred. The peptidescan be individual peptides, peptides co-administered with adjuvants,peptides coupled to peptides with different amino acid sequences and/orthe same peptides coupled to each other as repeating units. Also, whilepeptides may be directly coupled to each other, in some cases a smalllinker sequence or a larger heterolinker molecule may be advantageouslyused to couple the two peptides. For example, as the spacer, one or afew, up to about 5, preferably, up to about 3, neutral amino acids, suchas glycine, may be used to link the peptides. A preferred spacer peptideis GGG, however, the spacer may be made larger or smaller and altered toinclude other molecules besides the amino acid glycine. As examples ofheterolinkers may be made of, for example,N-succinimidyl-3-(2-pyridylthio)propinate (SPDP),m-maleimidobenzoyl-N-hydroxy-succimide (MBS) as well as any of the otherreagents employed to link peptides. When the peptides are not directlybonded the linking group will generally and preferably be any divalentlinking group. The linking group may be cleavable or non-cleavable underphysiological conditions or by appropriate inducement.

[0123] Although the total number of amino acids in the conjugatedpolypeptide is not particularly critical, from a practical aspect, theminimum number of amino acids, including any amino acid spacers orlinkers, will generally be at least about 10 or 12, preferably at leastabout 20, to obtain adequate antigen presentation and immunogenicity upto about 100 amino acids.

[0124] The polypeptides of this invention may be used as a vaccineeither prophylactically or therapeutically. When providedprophylactically the vaccine is provided in advance of any evidence ofmuscular contractile disorders. Antibodies are produced against thepeptides which are inotropic. The prophylactic administration of theinvention vaccine should serve to prevent or attenuate, for examplecardiac muscle contractile disorders. In a preferred embodiment a human,at high risk for heart muscle contractile disorders is prophylacticallytreated with a vaccine of this invention. When provided therapeutically,the vaccine is provided to enhance the patient's own antibody responseto produce the desired inotropic antibodies.

[0125] While it is possible for the immunogenic polypeptide, which mayor may not be conjugated, to be administered in a pure or substantiallypure form, it is preferable to present it as a pharmaceuticalcomposition, formulation or preparation.

[0126] The formulations of the present invention, both for clinical andfor human use, comprise a conjugated polypeptide as described above,together with one or more pharmaceutically acceptable carriers and,optionally, other therapeutic ingredients. The carrier(s) must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation and not deleterious to the recipient thereof. Theformulations may conveniently be presented in unit dosage form and maybe prepared by any method well-known in the pharmaceutical art.

[0127] In general, the formulations are prepared by uniformly andintimately bringing into association the active ingredient with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product into the desired formulation.

[0128] Formulations suitable for any route of administration may beused, such as, for example, intravenous, intramuscular, subcutaneous,intraperitoneal, nasal, oral, rectal, vaginal, etc. Generally, theformulations will comprise sterile aqueous solutions of the activeingredient with solutions which are preferably isotonic with the bloodof the recipient. Such formulations may be conveniently prepared bydissolving solid active ingredient in water containing physiologicallycompatible substances such as sodium chloride (e.g. 0.1-2.0M), glycine,and the like, and having a buffered pH compatible with physiologicalconditions to produce an aqueous solution, and rendering the solutionsterile. These may be present in unit or multi-dose containers, forexample, sealed ampoules or vials.

[0129] The formulations of the present invention may incorporate astabilizer. Illustrative stabilizers include polyethylene glycol,proteins, saccharides, amino acids, inorganic acids, and organic acidswhich may be used either on their own or as admixtures. Thesestabilizers, when used, are preferably incorporated in an amount ofabout 0.1 to about 10,000 parts by weight per part by weight ofimmunogen. If two or more stabilizers are to be used, their total amountis preferably within the range specified above. These stabilizers areused in aqueous solutions at the appropriate concentration and pH. Thespecific osmotic pressure of such aqueous solutions is generally in therange of about 0.1 to about 3.0 osmoles, preferably in the range ofabout 0.3 to about 1.2. The pH of the aqueous solution is adjusted to bewithin the range of about 5.0 to about 9.0, preferably within the rangeof 6-8. In formulating the immunogen of the present invention,anti-adsorption agent may be used.

[0130] Additional pharmaceutical methods may be employed to control theduration of action. Controlled release preparations may be achievedthrough the use of polymer to complex or absorb the conjugatedpolypeptide. The controlled delivery may be exercised by selectingappropriate macromolecules (for example polyester, polyamino acids,polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose,carboxymethylcellulose, or protamine sulfate) and the concentration ofmacromolecules as well as the methods of incorporation in order tocontrol release. Another possible method to control the duration ofaction by controlled-release preparations is to incorporate theconjugated polypeptide into particles of a polymeric material such aspolyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylenevinylacetate copolymers. Alternatively, instead of incorporating theseagents into polymeric particles, it is possible to entrap thesematerials in microcapsules prepared, for example, by coacervationtechniques or by interfacial polymerization, for example,hydroxy-methylcellulose or gelatin-microcapsules andpoly(methylmethacrylate) microcapsules, respectively, or in colloidaldrug delivery systems, for example, liposomes, albumin microspheres,microemulsions, nanoparticles, and nanocapsules or in macroemulsions.

[0131] When oral preparations are desired, the compositions may becombined with typical carriers, such as lactose, sucrose, starch, talc,magnesium stearate, crystalline cellulose, methyl cellulose,carboxymethyl cellulose, glycerin, sodium alginate or gum arabic amongothers. These carriers may likewise be used for preparing to beadministered via other cavities, e.g., nasal, rectal, etc.

[0132] The conjugated polypeptides of the present invention may besupplied in the form of a kit, alone, or in the form of a pharmaceuticalcomposition as described above.

[0133] As noted above, the administration of the vaccine of the presentinvention may be for either a prophylactic or therapeutic purpose. Whenprovided therapeutically, the immunogen is provided at (or after) theonset of the disease or at the onset of any symptom of the disease. Thetherapeutic administration of the immunogen serves to attenuate thedisease.

[0134] The present invention, therefore, provides antigenic conjugatedpolypeptides, which provide powerful vaccines for eliciting immuneresponses for production of inotropic antibodies in vivo.

[0135] The conjugated polypeptides, which may be prepared byconventional solid phase peptide synthesis or other conventional meansfor peptide synthesis, however, the peptides may also be prepared bygenetic engineering techniques. The DNA sequences coding for thepeptides of this invention can be prepared by any of the well knowntechniques for recombinant gene technology. For example, reference canbe made to the disclosure of recombinant proteins and peptides in U.S.Pat. No. 5,142,024 and the body of literature mentioned therein, thedisclosures of which are incorporated herein by reference thereto.

[0136] The following non-limiting examples are illustrative. Alldocuments mentioned herein are hereby incorporated by reference.

EXAMPLES

[0137] In the following examples, the following materials and methodswere employed.

[0138] Materials and Methods

[0139] Materials.

[0140] All reagents were purchased from Sigma Chemical Co., unlessspecified. Highly purified dog kidney (Na⁺+K⁺)-ATPase was a gift fromDr. Jack Kyte.

[0141] Antibody Preparation.

[0142] The RSATEEEPPNDD and DVEDSYGQQWTYEQR peptides were synthesizedaccording to the protein sequence reported (Schneider, J. W., Mercer, R.W., Caplan, M., Emanuel, J. R., Sweadner, K. J., Benz, E. J., Levenson,R. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 6357-6361; Xie, Z., Li, H.,Liu, G., Wang, Y., Askari, A., Mercer, R. W. (1994) Cloning of the dogNa/K-ATPase alpha 1 subunit. The Na Pump. (Bamberg, S., and Schoner, W.,Eds), pp. 49-52, Springer-Verlag, New York, N.Y.; Shull, M. M., Lingrel,J. B. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 4039-4043). Thepolyclonal Jianye antibody was generated in New Zealand White rabbitsusing KLH as a peptide carrier (Genemed). The immunoglobulins (IgG) werepurified through an affinity column directed against the same syntheticpeptide of the (Na⁺+K⁺)-ATPase. Purified antibodies recognize bothdenatured (by Western blots) and native (Na⁺+K⁺)-ATPase (byimmunocytostaining). Synthetic peptides were also utilized as thespecific peptide blockers for the antibodies.

[0143] Isolation of Cardiac Myocytes:

[0144] Ventricular cardiac myocytes were isolated from adultSprague-Dawley rats (2-3 months old; weight 225-300 g) using standardenzymatic techniques. Briefly, following anesthesia (sodiumpentobarbital, 100 mg/kg), the heart was quickly removed from the chestand aortic perfused at constant pressure (100 cmH₂O) at 37° C. for 3minutes with a Ca²⁺-free bicarbonate-based buffer containing 120 mMNaCl, 5.4 mM MgSO₄, 1.2 mM NaH₂PO₄, 5.6 mM glucose, 20 mM NaHCO₃, and 5mM taurine, in the presence of O₂ (95%)-CO₂ (5%). The enzymaticdigestion was initiated by adding collagenase (Worthington Type II, 1mg/ml) to the perfusion solution. Calcium (50 μM) was added to theenzyme solution when the heart became swollen. About 7 minutes later,the left ventricle was quickly removed, cut into several pieces, andfurther digested on a shaker (60-70 rpm) for 10 minutes in the sameenzyme solution. The supernatant containing the dispersed myocytes wasfiltered into a test tube and gently centrifuged at 500 rpm for 1minute. The cell pellet was then promptly resuspended in a solutioncontaining 0.125 M Ca²⁺. The supernatant was aspirated after themyocytes were pelleted by gravity for 10 minutes and the myocytes werethen resuspended in a solution containing 0.25 mM Ca²⁺. Theshake-harvest procedure was repeated several times until all of thepieces were digested. For freshly isolated cells, myocytes weresuspended in HEPES-buffer consisting of 1 mM CaCl₂, 0.137 mM NaCl, 5.4mM KCl, 15 mM dextrose, 1.3 mM MgSO₄, 1.2 mM NaH₂PO4, and 20 mM HEPES,pH 7.4.

[0145] Measurement of Mice Cardiac Contraction In Vivo:

[0146] Male wild-type mice (CD1, Charles River, 32-40 g) were utilizedfor this study. Mice In vivo cardiac functions were assessed bypressure-volume catheter in anesthetized mice. Briefly, mice wereinduced with 5% isofluorane, anesthetized with an intraperitonealinjection of urethane (300-500 mg/kg), etomidate (5 mg/kg) and morphine(0.5 mg kg-1), and intubated with a blunt 19G needle inserted viatracheostomy. Additional small dose was given when increase in heartrate or blood pressure was observed in response to tail pinch withforceps. Ventilation was initiated with 100% oxygen using acustom-designed, constant flow ventilator delivering a tidal volume of6.7 μL/kg at 120 breaths per min. The left external jugular vein wascannulated with a 30G needle connected to an infusion pump. Modestvolume expansion was provided (150 μL of 12.5% human albumin) at 50μL/min. Following stabilization, a lateral incision was made at thexyphoid cartilage to expose the left ventricular (LV) apex. The 1.4Fpressure-volume catheter (SPR-839, Millar Instruments Inc., Houston,Tex., USA) was inserted via an apical needle puncture with a 26G needle,and advanced along the cardiac long axis. A 2F pacing catheter (NuMed,Nicholville, N.Y., USA) was placed in the esophagus, dorsal to the leftatrium. Atrium was paced using 5-7 V, 2 ms pulses (SD25, GrassInstruments, Quincy, Mass., USA). Calibration of the volume signal wasperformed using a 5-10 μL bolus of 30% hypertonic saline injected intothe jugular vein to determine the signal offset and ultrasound flowprobe (AT01RB, Transonic Systems Inc.) placed around the thoracic aortato determine signal gain. Data were digitized at 2 kHz and stored todisk for off line analysis.

[0147] Mice cardiac atria pacing were maintained at constant beating(600 beats/min). The hearts were infused with PBS (5 μl/min) for 10 minprior for administration of SSA78 infusion (5 μl/min) for 30 minutes andfollowing by 10 min PBS washout at 5 μl/min. Control experiments showedthat the vehicle (PBS), at the experimental infusion rates, has noeffect on cardiovascular performance. Indices of myocardial systolic anddiastolic performance were derived from pressure-volume data obtainedboth at steady-state (every minute) and during transient loading of theheart with direct occlusion of the inferior vena cava (IVC) (every 5minutes). Steady-state indices were derived from 10 consecutive averagedbeats. Cardiac preload was indexed as the left ventricular end-diastolicvolume (EDV) and end-diastolic pressure (EDP). Cardiac afterload wasevaluated as effective arterial elastance (Ea; ratio of LV systolicpressure to stroke volume). This parameter is not preload dependent andhad been validated to closely approximate total afterload, whichincorporates systemic vascular resistance, aortic impedance, and thereflected wave properties of the vasculature. Myocardial contractilitywas indexed by cardiac output (CO), dPdt max, dPdt max normalized toinstantaneous developed pressure (dPdt max/IP), the load-independentend-systolic pressure-volume relationship (Ees) and preload recruitablestroke work (PRSW). Diastolic performance was measured by dPdt min andthe time constant of ventricular relaxation.

[0148] Measurement of Cell Contraction

[0149] Cardiac myocytes were isolated from wild-type adultSprague-Dawley rats, using a standard enzymatic method describedpreviously (Xu, K. Y., et al., (2002) BBRC, 289:167-172). Isolatedmyocytes were suspended in a buffer containing (in mM) 137 NaCl, 5.4KCl, 15 dextrose, 1.3 MgSO₄, 1.2 NaH₂PO₄, 1 CaCl₂, and 20 HEPES, pH 7.4.To measure cell contractility, cardiac myocytes were placed on aninverted microscope (Zeiss model IM-35), bathed with a HEPES-bufferedsolution, and electrically stimulated under 0.5 Hz at room temperature.The designated reagents were added when the baseline contraction wasstabilized after 10-15 min constant pacing. Cell length was monitoredfrom the bright-field image (650 nm to 750 nm red light illumination) byan optical edge-tracking method using a photodiode array (model 1024SAQ, Reticon) with a 3-ms time resolution. The contraction amplitude wasindexed by the percentage shortening of cell length.

[0150] Measurement of Intracellular Ca²⁺ Transients

[0151] Rat cardiac myocytes were loaded with 50 μg of a cell permeablefluorescent Ca²⁺ probe, Indo-1/acetoxymethyl-ester (Indo-1/AM, MolecularProbes, Inc., Eugene, Oreg.) for 10 min and then resuspended inHEPES-buffered solution in the presence of 1 mM Ca²⁺ and stored in thedark at room temperature for 60 min before be utilized for theexperiments. The Indo-1 loaded cells were placed on the stage of amodified inverted microscope (model IM-35; Carl Zeiss, Inc., Thornwood,N.Y.) equipped for simultaneous recording of Indo-1 flrorescence andcell length. Cells were electrically paced at 0.5 Hz at room temperatureand the excitation wavelength was selected by a 350-nm interferencefilter (bandwidth 10 nm; Oriel Corp., Stratford, Conn.). The ratio ofemission intensity at 410 nm to that at 490 nm was computed offline asan index of intracellular Ca²⁺ (Ca_(i) ²⁺) transient. The peak amplitudeof the transient is defined as the difference of the 410:490fluorescence ratio before and after electrical stimulation with orwithout antibody.

[0152] Isolation of Sarcolemmal Vesicles and Purification of(Na⁺+K⁺)-ATPase:

[0153] Rat cardiac sarcolemmal (SL) vesicles were isolated from ratheart muscle by sucrose flotation method. The vesicles were tested withsaponin and were predominately right-side-out in orientation.(Na⁺+K⁺)-ATPase was purified as described previously (Kyte, J. (1971)Biochemistry, 246:4157-4165). Briefly, the SL vesicles (4.4 mg/ml) weretitrated with 0.58 mg/ml of SDS in the presence of 2 mM ATP at 20° C.for 30 min. The SDS titrated fractions were then loaded on the top of asucrose (W/W) step gradient constructed with 10 ml of 37.3% (bottomstep), 20 ml of 28.8%, and 10 ml of 15% in a Ti 60 tube, and centrifugedat 40,000 rpm for 90 min. The fractions that contain (Na⁺+K⁺)-ATPase(between 37.3 and 28.8% on the sucrose gradient) were carefullycollected and sedimented at 40,000 rpm for 60 minutes. The purifiedenzyme was resuspended in a sucrose (250 mM)/histidinium chloride (30mM) buffer, pH 7.2, quick-frozen in liquid nitrogen and stored at 70° C.Highly purified dog kidney (Na⁺+K⁺)-ATPase was a gift from Dr. JackKyte.

[0154] Determination of (Na⁺+K⁺)-ATPase Activity:

[0155] The enzymatic activity was determined as described previously(Kyte J., et al., (1987) Biochemistry, 26:8350-8360) with modifications.Briefly, purified rat or dog (Na⁺+K⁺)-ATPase was incubated with orwithout Jianye or ouabain in the presence of 100 mM Na⁺ for 30 min atroom temperature. The reaction was initiated by adding 3 mM MgATP and 20mM K⁺ in a final volume of 0.25 ml at 37° C. for 30 min and terminatedby adding 0.75 ml quench solution and 0.025 ml developer. The color wasallowed to develop for 30 min at room temperature and the concentrationof phosphate was then determined at 700 nm using a spectrophotometer.

[0156] Echocardiography

[0157] Echocardiography (Echo) uses sound waves to form a picture of theheart valves and heart muscle. The Echo machine sends sound waves to atransducer (a sound sensitive instrument) that is placed on the animal'schest. The sound waves are reflected by the heart walls (muscle) andheart valves and back to the transducer, which changes the sound into apicture. Gel is applied on the rat chest and a transducer is placed overthe heart area. Heart structures are examined by changing the directionof the transducer. The sound waves cause no discomfort. Thus, an Echodetects the changes and provides information about heart chamber size,wall motion, valve movements, and structural changes in and around theheart.

Example 1 Jianye-2 Inotropic Antibody

[0158] Jianye-2 antibody, which specifically recognizes the RSATEEEPPNDDpeptide of the α-subunit of the (Na⁺+K⁺)-ATPase, has been found toincrease the contractility of isolated rat ventricular myocytes. FIG. 1shows the results obtained with immunofluorescent staining of Jianye-2antibody in rat cardiac myocytes and African green monkey CV-1 cells.Confocal image of rat cardiac myocytes in the presence of Jianye-2,shown in FIG. 1A or with both Jianye-2 antibody and peptide blocker(FIG. 1B). FIG. 1C shows the immunofluorescent stainings of Jianye-2antibody in a group of CV-1 cells at a magnification of 400×. FIG. 1Dshows the CV-1 cell image at 3000×. Jianye-2 antibody staining in thepresence of either 1 mM ouabain (FIG. 1E) or strophanthidin (FIG. 1F).The results reveal that ouabain and strophanthidin compete with theJianye-2 antibody binding site indicating that Jianye-2 antibodyspecifically binds to the (Na⁺+K⁺)-ATPase on the extracellular surfaceof the cell membrane. FIG. 2 shows results of the time courses of ratheart cell contraction with or without Jianye-2 antibody. Time runs fromleft to right. Column A, shows the baselines of rat heart cellcontraction. Columns B, C, D show the results obtained 10, 20, 30 minafter administration of either buffer (upper panel) or Jianye-2 antibody(lower panel, 85 nM). Enzyme activity was monitored in cell homogenatesunder the same experimental condition. The results show that Jianye-2antibody enhanced rat heart cell contraction without inhibiting(Na⁺+K⁺)-ATPase activity. FIG. 3 shows that Jianye-2 antibody markedlyincreased intracellular Ca²⁺ contraction and demonstrates thatintracellular Ca² ⁺ concentration is involved in the mechanisms ofJianye-2 antibody enhanced heart cell contraction. The data represent amean of 12 independent experiments. FIG. 4 shows the dose-dependentcontractile response of Jianye-2 antibody in rat ventricular myocytes.The half maximal contractile response (EC50) is 35 nM. The datarepresent a mean of four independent experiments.

[0159]FIG. 5 is a schematic representation of pressure-volume loops ofthe effect of Jianye-2 antibody on normal mouse heart left ventricle.Jianye-2 antibody (total 150 ml, 6.0 μM in PBS, pH 7.2) was administeredto the mouse heart at a rate of 5 ml/min. The results show that Jianye-2antibody dramatically induced a reversible positive inotropic effect asdemonstrated by the changes in ventricular pressure-volume loops withtime during the cardiac cycle. The results represent one of the sevensimilar experiments. FIG. 6 shows the time- and concentration-dependentJianye-2 antibody effects on mouse heart contraction in vivo model. Atdifferent fixed concentrations of Jianye-2 antibody as indicated in thefigure, the data are plotted in the form of percent of mouse heartcontraction as a function of time. Compared with the background control(at 0 min as 100%), Jianye-2 antibody increased mouse heart contractionsare expressed as percentages (%) for 5, 10, 15, and 20 min respectivelyafter administration of 3.4 mM (open circles) or 6.0 mM (black circles)of Jianye-2 antibody. Mouse heart contraction was 108(±5.3), 113(±7),116(±10), 122(±9.0) as shown in the open circles, and 115(±9.0),127(±21), 137(±16), and 143(±30) in black circles. First order rateconstants were 1.06 and 2.16%/min for 3.4 mM and 6.0 mM Jianye-2antibody, respectively. The data represent the mean of 5 independentdeterminations.

Example 2 KX-1 Inotropic Antibody

[0160] Antibody (KX-1), which recognizes DVEDSYGQQWTYEQR peptide ofα-subunit of the (Na⁺+K⁺)-ATPase, has been found to increase thecontractility of isolated rat heart cells. Vaccination of its specificpeptide-antigen in heart diseased animal model significantly decreasedthe progression of heart failure. This KX-1 antibody, its peptidevaccine, and monoclonal and humanized antibodies are useful fortreatment of heart failure and other contractile disorders. FIG. 7 showsthat the KX-1 antibody enhanced the velocity of shortening of ratventricular myocyte and increased the force of contraction of the heartcells. The results indicate that the KX-1 antibody is an inotropicagent.

[0161]FIG. 8 represents the changes of intracellular calcium transientfollowing the binding of KX-1 to the (Na⁺+K⁺)-ATPase and indicates thatKX-1 induced heart cell contraction is dependent on intracellular Ca²⁺increase. FIG. 9 reveals that the DVEDSYGQQWTYEQR peptide can beutilized as a vaccine to generation of endogenous KX-1 antibody in heartfailure rats and the results show that endogenous KX-1 antibody delayedthe rate of progression of heart failure in live heart failure ratanimal models. In contrast, cardiac function was significantly depressedin the control heart failure rat without immunization with KX-1 antigen.

What is claimed:
 1. An antibody which recognizes the amino acid sequencecomprising RSATEEEPPNDD of the α-subunit of (Na⁺+K⁺)-ATPase enzyme, oran isoform of the amino acid sequence.
 2. The antibody of claim 1,wherein binding of the antibody to the amino acid sequence,RSATEEEPPNDD, of the α-subunit of (Na⁺+K⁺)-ATPase increases myocyteintracellular diastolic and systolic calcium.
 3. The antibody of claim1, wherein binding of the antibody to the amino acid sequence,RSATEEEPPNDD, of the α-subunit of (Na⁺+K⁺)-ATPase exerts a positiveinotropic effect in cardiac myocytes.
 4. The antibody of claim 1,wherein the antibody is a polyclonal antibody.
 5. The antibody of claim1, wherein the antibody is a monoclonal antibody.
 6. The antibody ofclaim 1, wherein the antibody is a humanized antibody.
 7. The antibodyof claim 1, wherein the antibody is administered to a patient in aneffective therapeutic amount to treat the patient suffering from orsusceptible to heart disease and/or muscle contractile disorders.
 8. Anantibody which recognizes the amino acid sequence comprisingDVEDSYGQQWTYEQR of the α-subunit of (Na⁺+K⁺)-ATPase enzyme, of whichrecognizes an isoform of the amino acid sequence.
 9. The antibody ofclaim 8, wherein binding of the antibody to the amino acid sequence,DVEDSYGQQWTYEQR, of the α-subunit of (Na⁺+K⁺)-ATPase increases myocyteintracellular diastolic and systolic calcium.
 10. The antibody of claim8, wherein binding of the antibody to the amino acid sequence,DVEDSYGQQWTYEQR, of the α-subunit of (Na⁺+K⁺)-ATPase exerts a positiveinotropic effect in cardiac myocytes.
 11. The antibody of claim 8,wherein the antibody is a polyclonal antibody.
 12. The antibody of claim8, wherein the antibody is a monoclonal antibody.
 13. The antibody ofclaim 8, wherein the antibody is a humanized antibody.
 14. The antibodyof claim 8, wherein the antibody is administered to a patient in aneffective therapeutic amount to treat the patient suffering from orsusceptible to heart disease and/or muscle contractile disorders.
 15. Apurified peptide comprising the amino acid sequence RSATEEEPPNDD orderivatives or isoform thereof.
 16. The peptide of claim 15, wherein thepeptides are administered individually or in combination in apharmaceutically acceptable carrier to a patient.
 17. A nucleic acidvector encoding an amino acid sequence comprising RSATEEEPPNDD orisoform thereof.
 18. The vector of claim 17, wherein the vectorcomprises tissue specific promoters.
 19. The vector of claim 17, whereinthe tissue specific promoters are cardiac tissue specific.
 20. Thevector of claim 17, wherein the in vivo generated antibodies bind to theamino acid sequence, RSATEEEPPNDD, of the α-subunit of (Na⁺+K⁺)-ATPase.21. The vector of claim 18, wherein binding of the in vivo generatedantibodies to the amino acid sequence, RSATEEEPPNDD, of the α-subunit of(Na⁺+K⁺)-ATPase increases myocyte intracellular diastolic and systoliccalcium.
 22. The vector of claim 18, wherein binding of the in vivogenerated antibodies to the amino acid sequence, RSATEEEPPNDD, of theα-subunit of (Na⁺+K⁺)-ATPase exerts a positive inotropic effect incardiac myocytes.
 23. The vector of claim 18, wherein the vector isadministered to a patient in an effective therapeutic amount to treatthe patient suffering from or susceptible to heart disease and/or musclecontractile disorders.
 24. A purified peptide comprising the amino acidsequence DVEDSYGQQWTYEQR or derivative or isoform thereof.
 25. Thepeptide of claim 24, wherein the peptides are administered individuallyor in combination in a pharmaceutically acceptable carrier to a patient.26. A nucleic acid vector encoding an amino acid sequence comprisingDVEDSYGQQWTYEQR.
 27. The vector of claim 26, wherein the vectorcomprises tissue specific promoters.
 28. The vector of claim 26, whereinthe tissue specific promoters are cardiac tissue specific.
 29. Thevector of claim 26, wherein the in vivo generated antibodies bind to theamino acid sequence, DVEDSYGQQWTYEQR, of the α-subunit of(Na⁺+K⁺)-ATPase.
 30. The vector of claim 27, wherein binding of the invivo generated antibodies to the amino acid sequence, DVEDSYGQQWTYEQR,of the α-subunit of (Na⁺+K⁺)-ATPase increases myocyte intracellulardiastolic and systolic calcium.
 31. The vector of claim 27, whereinbinding of the in vivo generated antibodies to the amino acid sequence,DVEDSYGQQWTYEQR, of the α-subunit of (Na⁺+K⁺)-ATPase exerts a positiveinotropic effect in cardiac myocytes.
 32. The vector of claim 27,wherein the vector is administered to a patient in an effectivetherapeutic amount to treat the patient suffering from or susceptible toheart disease and/or muscle contractile disorders.
 33. A method ofgenerating antibodies, wherein binding of the antibodies to an epitopeof the α-subunit of (Na⁺+K⁺)-ATPase exerts a positive inotropic effectin cardiac myocytes, comprising: generating amino acid sequencescorresponding to overlapping peptide fragments of the α-subunit of(Na⁺+K⁺)-ATPase and variants thereof; and, obtaining antibodies specificfor each peptide fragment by standard methods; and, determining theeffects of the antibodies on intracellular diastolic and systoliccalcium levels and cell shortenings as compared to controls.
 34. Themethod of claim 33, wherein binding of the antibodies to the α-subunitof (Na⁺+K⁺)-ATPase exerts a positive inotropic effect in cardiacmyocytes.
 35. The method of claim 34, wherein binding of the antibodiesto the α-subunit of (Na⁺+K⁺)-ATPase increases myocyte intracellulardiastolic and systolic calcium.
 36. The method of claim 35, whereinbinding of the antibodies to the α-subunit of (Na⁺+K⁺)-ATPase increasesmyocytes contractions as compared to controls.
 37. The method of claim34, wherein the antibodies generated are polyclonal antibodies.
 38. Themethod of claim 34, wherein the antibodies generated are monoclonalantibodies.
 39. The method of claim 34, wherein the antibody isadministered to a patient in an effective therapeutic amount to treatthe patient suffering from or susceptible to heart disease and/or musclecontractile disorders.
 40. The method of claim 34, wherein the antibodyis administered to a patient in a therapeutically effective amount toblock other molecules from binding to drug-interaction sites of(Na⁺+K⁺)-ATPase, wherein the patient is suffering from or susceptible toarhythmias, tachyrhithmias and the like.
 41. The antibodies of claim 40,wherein the antibodies eliminate negative inotropic agents.
 42. A methodfor diagnosing heart failure and/or contractile disorders comprising:isolating heart tissue; and, allowing the binding of inotropicantibodies to epitopes of isolated heart tissue; and, measuringintracellular diastolic and systolic calcium and cell shortenings. 43.The method of claim 42, wherein inotropic antibodies binding to epitopesof the α-subunit of (Na⁺+K⁺)-ATPase from cells of patients sufferingfrom or susceptible to heart failure and/or contractile disorders willhave a lower inotropic effect as compared to healthy individuals.
 44. Amethod for targeting and blocking the RSATEEEPPNDD site of α-subunit ofthe (Na⁺+K⁺)-ATPase, comprising: contacting a myocyte with a desiredmolecule; and, measuring the intracellular diastolic and systolic Ca²⁺;and, measuring cell shortening and heart function; whereby, identifyingmolecules useful for therapy of patients suffering from or susceptibleto heart disease and other contractile disorders.
 45. The method ofclaim 44, wherein the desired molecules are administered to patientssuffering from and/or susceptible to heart disease and other contractiledisorders, an effective therapeutic amount of desired molecules.
 46. Amethod for targeting and blocking the DVEDSYGQQWTYEQR site of α-subunitof the (Na⁺+K⁺)-ATPase, comprising: contacting a myocyte with a desiredmolecule; and, measuring the intracellular diastolic and systolic Ca²⁺;and, measuring cell shortening and heart function; whereby, identifyingmolecules useful for therapy of patients suffering from or susceptibleto heart disease and other contractile disorders.
 47. The method ofclaim 46, wherein the desired molecules are administered to patientssuffering from and/or susceptible to heart disease and other contractiledisorders, an effective therapeutic amount of desired molecules.